Endothelial Cells Group Research Articles

Comparison the therapeutic effects of bone marrow CD144+ endothelial cells and CD146+ mesenchymal stem cells in POF rats.

Premature ovarian insufficiency (POI) is a challenging issue in terms of reproduction biology. In this study, therapeutic properties of bone marrow CD146+ mesenchymal stem cells (MSCs) and CD144+ endothelial cells (ECs) were separately investigated in rats with POI. POI rats were classified into control POI, POI + CD146+ MSCs, and POI + CD144+ ECs groups. Enriched CD146+ MSCs and CD144+ ECs were directly injected into ovarian tissue (15 × 104 cells/10 μL) in relevant groups. After 4 weeks, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol (E2) levels were measured in blood samples. Ovarian tissues were collected and subjected to Hematoxylin-Eosin and Masson's trichrome staining. The expression of angp-2, vegfr-2, smad-2, -4, -6, and tgf-β1 was studied using qRT-PCR analysis. Histopathological examination indicated an increased pattern of atretic follicles in the POI group related to the control rats (P<0.0001). Data indicated that injection of POI + CD146+ MSCs and CD144+ ECs in POI rats reduced atretic follicles and increased the number of normal follicles (P<0.01). Along with these changes, the content of blue-colored collagen fibers was diminished after cell transplantation. Besides, cell transplantation in POI rats had the potential to reduce increased FSH, and LH levels (P<0.05). In contrast, E2 content was increased in POI + CD146+ MSCs and POI + CD144+ ECs groups compared to control POI rats, indicating restoration of follicular function. CD144+ (smad-2, and -4) and CD146+ (smad-6) cells altered the activity of genes belonging TGF-β signaling pathway. Unlike POI + CD146+ MSCs, aberrant angiogenesis properties were significantly down-regulated in POI + CD144+ ECs related to the control POI group (P<0.05). The transplantation of bone marrow CD146+ and CD144+ cells can lead to the restoration of ovarian tissue function in POI rats via modulating different mechanisms associated with angiogenesis and fibrosis.

Engineered multi-functional, pro-angiogenic collagen-based scaffolds loaded with endothelial cells promote large deep burn wound healing.

The lack of vascularization associated with deep burns delays the construction of wound beds, increases the risks of infection, and leads to the formation of hypertrophic scars or disfigurement. To address this challenge, we have fabricated a multi-functional pro-angiogenic molecule by grafting integrin αvβ3 ligand LXW7 and collagen-binding peptide (SILY) to a dermatan sulfate (DS) glycosaminoglycan backbone, named LXW7-DS-SILY (LDS), and further employed this to functionalize collagen-based Integra scaffolds. Using a large deep burn wound model in C57/BLK6 mice (8-10weeks old, 26-32g, n = 39), we demonstrated that LDS-modified collagen-based Integra scaffolds loaded with endothelial cells (ECs) accelerate wound healing rate, re-epithelialization, vascularization, and collagen deposition. Specifically, a 2 cm × 3cm full-thickness skin burn wound was created 48h after the burn, and then wounds were treated with four groups of different dressing scaffolds, including Integra + ECs, Integra + LDS, and Integra + LDS + ECs with Integra-only as the control. Digital photos were taken for wound healing measurement on post-treatment days 1, 7, 14, 21, 28, and 35. Post-treatment photos revealed that treatment with the Intgera + LDS + ECs scaffold exhibited a higher wound healing rate in the proliferation phase. Histology results showed significantly increased re-epithelialization, increased collagen deposition, increased thin and mixed collagen fiber content, increased angiogenesis, and shorter wound length within the Integra + LDS + ECs group at Day 35. On Day 14, the Integra + LDS + ECs group showed the same trend. The relative proportions of collagen changed from Day 14 to Day 35 in the Integra + LDS + ECs and Integra + ECs groups demonstrated decreased thick collagen fiber deposition and greater thin and mixed collagen fiber deposition. LDS-modified Integra scaffolds represent a promising novel treatment to accelerate deep burn wound healing, thereby potentially reducing the morbidity associated with open burn wounds. These scaffolds can also potentially reduce the need for autografting and morbidity in patients with already limited areas of harvestable skin.

Endoderm-derived islet1-expressing cells differentiate into endothelial cells to function as the vascular HSPC niche in zebrafish.

Endothelial cells (ECs) line blood vessels and serve as a niche for hematopoietic stem and progenitor cells (HSPCs). Recent data point to tissue-specific EC specialization as well as heterogeneity; however, it remains unclear how ECs acquire these properties. Here, by combining live-imaging-based lineage-tracing and single-cell transcriptomics in zebrafish embryos, we identify an unexpected origin for part of the vascular HSPC niche. We find that islet1 (isl1)-expressing cells are the progenitors of the venous ECs that constitute the majority of the HSPC niche. These isl1-expressing cells surprisingly originate from the endoderm and differentiate into ECs in a process dependent on Bmp-Smad signaling and subsequently requiring npas4l (cloche) function. Single-cell RNA sequencing analyses show that isl1-derived ECs express a set of genes that reflect their distinct origin. This study demonstrates that endothelial specialization in the HSPC niche is determined at least in part by the origin of the ECs.

Biological activity of a vascular endothelial cell-hydroxyapatite orbital implant complex: An experimental study.

The reduction in postoperative complications is a considerable concern after orbital reparation and reconstruction. Selecting the appropriate scaffold materials to improve the survival rates of the seeded cells is a challenge in tissue engineering. The aim of the present study was to evaluate the biological activity of a vascular endothelial cell-hydroxyapatite orbital complex, which was constructed with tissue engineering and used as an implant after enucleation of the eyeNew Zealand white rabbits were randomly divided into two groups that underwent hydroxyapatite orbital implant surgery in the right eye. The primary orbital microvascular endothelial cells were collected from the microvascular tissue and subsequently cultured. Then, hydroxyapatite ocular implants were cultured with vascular endothelial cells in the endothelial cell (EC) group, and implants were cultured without vascular endothelial cells in the blank group. Characterization of the cells was performed with immunofluorescence staining and a Transwell migration and cell tube formation assay. The levels of interleukin-8 (IL-8) and vascular endothelial growth factor (VEGF) in the rabbit conjunctiva were measured with an ELISA. The results showed that the levels of IL-8 were decreased in the EC group and increased in the blank group. The levels of VEGF were increased in the EC group when compared to the blank group with statistical significance. The average depth of the fibrovascular tissue was obviously thicker in the EC group compared with that found in the blank group. These findings suggest that the vascular endothelial cell-hydroxyapatite orbital implant complex may be an effective strategy with which to accelerate vascularization and reduce complications of infection with satisfactory biological activity.

Effect of microvascular pericytes of cochlear stria vascularis on endothelial cell permeability in C57BL/6J mice

Objective: To study the changes in the permeability of the blood labyrinth barrier of the aging cochlea in mice, and to establish a non-contact co-culture model of endothelial cells (EC) and pericytes (PC) to furtherly investigate the cochlear stria vascularis microvascular pericytes impact on the permeability of endothelial cells. Methods: C57BL/6J mice were divided into two groups, three months old as young group, 12 months old as senile group. Cell experiment was divided into four groups, EC group, EC+PC co-culture group, D-gal+EC group and D-gal+EC+PC co-culture group. Auditory brainstem response (auditory brain response, ABR) was used to detect the auditory function of the two groups of mice. Evans blue staining was applied to detect the permeability of the cochlear blood labyrinth barrier of the two groups of mice. Transmission electron microscopy was used to observe the ultrastructure of blood labyrinth barrier endothelial cells, pericytes and tight junctions in the two groups of mice. Immunohistochemistry was used to detect the expression levels of tight junction proteins in the stria vascularis of the cochlea of the two groups of mice. Transwell chamber was used to detect the permeability of endothelial cells. Western blot and immunofluorescence technology were used to detect the expression level of tight junction protein on endothelial cells. SPSS 20.0 software was used to analyze the data. Results: Compared with the young group, the ABR threshold of the aging group was significantly increased, the latency of wave I was prolonged (t=10.25, P<0.01;t=5.61, P<0.05), the permeability of the cochlear blood labyrinth barrier was increased and the expression of tight junction protein on the vascular stria was decreased (P<0.05). The cochlear ultrastructure showed that the cochlear vascular stria microvascular lumen was deformed, the basement membrane thickened and the tight junction gap between endothelium enlarged. The positive rate of ECs and PCs in primary culture was more than 95%. The cells induced by 15 g/L D-gal were determined to be senescent cells. Compared with EC group, the expression of tight junction protein in endothelial cells of D-gal+EC group decreased（t=7.42，P<0.01；t=13.19，P<0.05）and the permeability increased (t=11.17, P<0.01). In the co-culture group, the expression of tight junction protein between endothelial cells in EC+PC co-culture group and D-gal+EC+PC co-culture group increased and the permeability decreased. Conclusions: In aging mice, the permeability of cochlear blood labyrinth barrier will increase and the level of tight junction protein will decrease; in aging state, cochlear vascular stria microvascular pericytes may affect endothelial cell permeability by regulating the expression of tight junction protein.

Effects of adipose-derived stem cells and endothelial cells on survival and neovascularization of fat tissue transplants

To investigate the effects of adipose-derived stem cells (ADSCs) and endothelial cells (ECs) on the survival and neovascularization of fat tissue transplants. The ADSCs were isolated by collagenase digestion from the adipose tissues voluntarily donated by the patients undergoing mastectomy, and subcultured. The passage 3 ADSCs were used for subsequent experiments. The residual fat tissues were used to prepare fat particles (FPs). The human umbilical vein endothelial cells (HUVECs) were used as ECs for subsequent experiments. Eighty healthy male nude mice, aged 4-6 weeks, were randomly divided into 4 groups ( n=20). The mice were received subcutaneous injection at the dorsum of 1 mL FPs+0.3 mL normal saline (NS) in control group, 1 mL FPs+2×10 6 ECs+0.3 mL NS in ECs group, 1 mL FPs+2×10 6 ADSCs+0.3 mL NS in ADSCs group, and 1 mL FPs+1×10 6 ECs+1×10 6 ADSCs+0.3 NS in ADSCs+ECs group. General observations of the injection sites were performed, and the survival of the mice was recorded. At 2, 4, 8, and 12 weeks after injection, grafted fat tissues were firstly assessed by ultrasonography, then they were collected for volume measurement (water displacement method) and histology observation (HE staining and immunofluorescence staining). All mice survived until the end of experiment. At each time point, no significant difference was noted between groups in ultrasonography assay. There was no significant blood flow signal in the grafted fat tissues, or cysts, calcification, solid occupying in recipient area. Generally, the volume of grafted fat tissues decreased with time in all groups. Specifically, the volumes of grafted fat tissues were larger in ADSCs group and ADSCs+ECs group than that in control group and ECs group ( P<0.05) at each time point, and in ADSCs group than in ADSCs+ECs group ( P<0.05) at 8 and 12 weeks. HE staining showed that all groups had similar tendencies in general histology changes, and remodeling in ADSCs group was the fastest than in the other groups. By immunofluorescence staining for neovascularization, the new vessels in all groups were increasing with time. The vessel densities were higher in ECs group, ADSCs group, and ADSCs+ECs group than in control group ( P<0.05) at each time point, in ADSCs group than in ECs group and ADSCs+ECs group ( P<0.05) at 4 weeks, in ADSCs group and ADSCs+ECs group than in ECs group ( P<0.05) at 8 and 12 weeks. ADSCs can significantly increase the survival of transplanted fat tissue, which may be related to promoting the neovascularization.

Retinal vascular injuries and intravitreal human embryonic stem cell-derived haemangioblasts.

To investigate whether intravitreally applied haemangioblasts (HB) derived from human embryonic stem cells (hESCs) are helpful for the repair of vascular damage caused in animals by an oxygen-induced retinopathy (OIR), by an induced diabetic retinopathy (DR) or by an induced retinal ischaemia with subsequent reperfusion. Human embryonic stem cell-derived HBs were transplanted intravitreally into C57BL/6J mice (OIR model), into male Wistar rats with an induced DR and into male Wistar rats undergoing induced retinal ischaemia with subsequent reperfusion. Control groups of animals received an intravitreal injection of endothelial cells (ECs) or phosphate-buffered saline (PBS). We examined the vasculature integrity in the mice with OIR, the blood-retina barrier in the rats with induced DR, and retinal thickness and retinal ganglion cell density in retina flat mounts of the rats with the retinal ischaemic-reperfusion retinopathy. In the OIR model, the study group versus control groups showed a significantly (p<0.001) smaller retinal avascular area [5.1±2.7%;n=18 animals versus 12.2±2.8% (PBS group; n=10 animals) and versus 11.8±3.7% (EC group; n=8 animals)] and less retinal neovascularization [6.3±2.5%;n=18 versus 15.2±6.3% (n=10; PBS group) and versus 15.8±3.3% (n=8; EC group)]. On retinal flat mounts, hESC-HBs were integrated into damaged retinal vessels and stained positive for PECAM (CD31) as EC marker. In the DR model, the study group versus the EC control group showed a significantly (p=0.001) better blood-retina barrier function as measured at 2days after the intravitreal injections [study group: 20.2±12.8μl/(g×hr); n=6; versus EC control group: 52.9±9.9μl/(g×hr; n=6)]. In the retinal ischaemia-reperfusion model, the groups did not differ significantly in retinal thickness and retinal ganglion cell density at 2, 5 and 7days after baseline. By integrating into damaged retinal vessels and differentiating into ECs, intravitreally administered hESC-HBs may have partially repaired a retinal vascular injury caused by OIR model and DR.

Angiogenic and Immunomodulatory Properties of Endothelial and Mesenchymal Stem Cells.

It has been suggested that the effect of implanted cells on the local environment is important when selecting the appropriate cell type for tissue regeneration. Our aim was to compare the local tissue response to implanted human mesenchymal stem cells (MSC) and human umbilical vein endothelial cells (EC). MSC and EC were cultured in poly(l-lactide-co-1,5-dioxepan-2-one) scaffolds for 1 week in a bioreactor system, after which they were implanted subcutaneously in NOD/SCID mice. After 3 weeks, scaffolds were retrieved, and the mRNA expression of selected genes involved in hypoxia and inflammation was examined by real-time reverse transcription polymerase chain reaction and correlated with immunofluorescent staining for corresponding proteins. The Toll-like receptor signaling pathway was examined by superarray hybridization. The expression of 53 angiogenesis-related proteins was investigated by a proteome profiler angiogenesis antibody array kit. Vascularization was quantified using immunohistochemistry for CD31. The expression of hypoxia-inducible factors and biomarkers for angiogenesis was more strongly upregulated in response to implanted EC than to MSC, suggesting a higher sensitivity to low oxygen tension among EC. Hypoxic signaling was increased after implantation of EC compared with MSC, leading to a prolonged acute inflammatory phase that promoted ingrowth of vascular cells and establishment of the circulation. Inflammatory cytokines were also differently expressed at the gene and protein levels in the two experimental groups, resulting in altered recruitment of acute and chronic inflammatory cells. The end result of these differences was increased vessel formation within the constructs in the EC group.

Biofabrication enables efficient interrogation and optimization of sequential culture of endothelial cells, fibroblasts and cardiomyocytes for formation of vascular cords in cardiac tissue engineering

We previously reported that preculture of fibroblasts (FBs) and endothelial cells (ECs) prior to cardiomyocytes (CMs) improved the structural and functional properties of engineered cardiac tissue compared to culture of CMs alone or co-culture of all three cell types. However, these approaches did not result in formation of capillary-like cords, which are precursors to vascularization in vivo. Here we hypothesized that seeding the ECs first on Matrigel and then FBs 24 h later to stabilize the endothelial network (sequential preculture) would enhance cord formation in engineered cardiac organoids. Three sequential preculture groups were tested by seeding ECs (D4T line) at 8%, 15% and 31% of the total cell number on Matrigel-coated microchannels and incubating for 24 h. Cardiac FBs were then seeded (32%, 25% and 9% of the total cell number, respectively) and incubated an additional 24 h. Finally, neonatal rat CMs (60% of the total cell number) were added and the organoids were cultivated for seven days. Within 24 h, the 8% EC group formed elongated cords which eventually developed into beating cylindrical organoids, while the 15% and 31% EC groups proliferated into flat EC monolayers with poor viability. Excitation threshold (ET) in the 8% EC group (3.4 ± 1.2 V cm−1) was comparable to that of the CM group (3.3 ± 1.4 V cm−1). The ET worsened with increasing EC seeding density (15% EC: 4.4 ± 1.5 V cm−1; 31% EC: 4.9 ± 1.5 V cm−1). Thus, sequential preculture promoted vascular cord formation and enhanced architecture and function of engineered heart tissues.

Spin-Labeling Magnetic Resonance Imaging Detects Increased Myocardial Blood Flow After Endothelial Cell Transplantation in the Infarcted Heart

We quantified absolute myocardial blood flow (MBF) using a spin-labeling MRI (SL-MRI) method after transplantation of endothelial cells (ECs) into the infarcted heart. Our aims were to study the temporal changes in MBF in response to EC transplantation and to compare regional MBF with contractile function (wall motion) and microvascular density. We first validated the SL-MRI method with the standard microsphere technique in normal rats. We then induced myocardial infarction in athymic rats and injected 5 million ECs (human umbilical vein endothelial cells) suspended in Matrigel or Matrigel alone (vehicle) along the border of the blanched infarcted area. At 2 weeks after myocardial infarction, MBF averaged over the entire slice (P=0.038) and in the infarcted region (P=0.0086) was significantly higher in EC versus vehicle group; the greater MBF was accompanied by an increase of microvasculature density in the infarcted region (P=0.0105 versus vehicle). At 4 weeks after myocardial infarction, MBF in the remote region was significantly elevated in EC-treated hearts (P=0.0277); this was accompanied by increased wall motion in this region assessed by circumferential strains (P=0.0075). Intraclass correlation coefficients and Bland-Altman plot revealed a good reproducibility of the SL-MRI method. MBF in free-breathing rats measured by SL-MRI is validated by the standard color microsphere technique. SL-MRI allows quantification of temporal changes of regional MBF in response to EC treatment. The proof-of-principle study indicates that MBF is a unique and sensitive index to evaluate EC-mediated therapy for the infarcted heart.

Vascular therapy for radiation cystitis

The underlying pathology of radiation cystitis is cellular and vascular damage followed by increased fibrosis and inflammation. This study was to determine if neovascular-promoting therapy could reduce the pathological changes in the bladder wall associated with pelvic irradiation. Adult female Lewis inbred rats were irradiated with a single dose of 20 Gy directed at their bladder. Four weeks later, 30 rats were divided equally into one of three treatment groups for bladder wall injection of: (1) PBS (Control); (2) PBS containing 50 ng vascular endothelial growth factor (VEGF (165)); or (3) PBS containing 1 × 10(6) rat endothelial cells (EC). Age-matched non-irradiated rats (n = 10) served as untreated controls. At either 1.5 or 3 months following radiation, bladders were analyzed for collagen deposition using Masson's Trichrome staining of collagen and muscle and vascularization using Von Willebrand factor staining of ECs. Quantitative-PCR was used to examine markers of angiogenesis, hypoxia, and fibrosis. The collagen/muscle ratio was doubled in the control group 3 months post-irradiation (P < 0.05 vs. non-irradiated bladders). Both ECs and VEGF inhibited increases in collagen content (P < 0.05 vs. control). Similarly, irradiation reduced bladder wall vessel counts compared to non-irradiated controls (P < 0.05) and both ECs and VEGF maintained vessel counts similar to that of non-irradiated controls (P < 0.05). PCR analysis showed a higher expression of neovascular markers (CD31, KDR) in the EC and VEGF groups compared to non-irradiated controls (P < 0.05). Angiogenesis therapy may be useful in the prevention and/or treatment of the underlying pathology of radiation cystitis.

Pressure shift mediated anoikis of endothelial cells in the flow field in vitro

Dramatic changes of pressure in the local circulation flow field would lead to alterations in biorheological characteristics of Endothelial cells(ECs), and futher resulted in the apoptosis induced by loss of anchorage, a form of cell death known as anoikis. In this study, we set levels of pressure(negative and positive pres- sure) loaded ECs groups and non-activated cultured ECs ,single shear stress loaded ECs as control group to demonstrate the effects of pressure shift on cell morphogenesis and adhesion. Furthermore, we in- vestigate the effects of pressure shift on ECs proli- feration and apoptosis to elucidate the influences of pressure shift on vitality of ECs. We present these data here to suggest that the negative pressure might be another important factor beyond velocity and shear stress in biomechanical impairment on ECs, then to trigger the apoptosis with the extracellular matrix (ECM) detachment (anoikis). As the negative pressure is thought to play a role in the anoikis process, these results have implications for both the path- ogenesis and therapeutics investigations of stenostic vessel dis- eases and the future vascular tissue engineering.

Improved Islet Survival and Funtion With Rat Endothelial Cells In Vitro Co-Culture

Objective Pancreatic islet transplantation is an emerging therapy for type 1 diabetes. To preserve its function, transplanted islets must be revascularized because arterial and venous connections are disrupted during islet isolation. The current paradigm is that islet revascularization originates from the transplant recipient. This study was designed to test whether the function of isolated islets can be retained by co-culture with thoracic aorta endothelial cells in vitro. Methods Sprague-Dawley rats were used in this study. The endothelial cells (ECs) were isolated from the thoracic aorta. The viability of the isolated islets was assessed by acridine orange/propidium iodide (AO/PI) double staining. The islets were either placed in standard cultures (group A) or in co-cultures with ECs (group B). Islet viablity was assessed by an insulin release assay. Results The islets in group B exhibited normal morphology with >90% staining positive as detected by AO/PI with 7 days. Insulin release assays showed a significantly higher simulation index (SI) in group B compared with group A ( P < .05) except on the first day. Conclusion This study suggested that co-cultrue of freshly isolated rat islets with ECs improves postculture survival and islet function in vitro.

Engineering angiogenesis following spinal cord injury: a coculture of neural progenitor and endothelial cells in a degradable polymer implant leads to an increase in vessel density and formation of the blood-spinal cord barrier.

Angiogenesis precedes recovery following spinal cord injury and its extent correlates with neural regeneration, suggesting that angiogenesis may play a role in repair. An important precondition for studying the role of angiogenesis is the ability to induce it in a controlled manner. Previously, we showed that a coculture of endothelial cells (ECs) and neural progenitor cells (NPCs) promoted the formation of stable tubes in vitro and stable, functional vascular networks in vivo in a subcutaneous model. We sought to test whether a similar coculture would lead to the formation of stable functional vessels in the spinal cord following injury. We created microvascular networks in a biodegradable two-component implant system and tested the ability of the coculture or controls (lesion control, implant alone, implant + ECs or implant + NPCs) to promote angiogenesis in a rat hemisection model of spinal cord injury. The coculture implant led to a fourfold increase in functional vessels compared with the lesion control, implant alone or implant + NPCs groups and a twofold increase in functional vessels over the implant + ECs group. Furthermore, half of the vessels in the coculture implant exhibited positive staining for the endothelial barrier antigen, a marker for the formation of the blood-spinal cord barrier. No other groups have shown positive staining for the blood-spinal cord barrier in the injury epicenter. This work provides a novel method to induce angiogenesis following spinal cord injury and a foundation for studying its role in repair.

Sexual and Maturational Difference in Phosphodiesterase mRNA Expression in Rat Skeletal Muscle Microvascular Endothelial Cells

The levels of intracellular cAMP and cGMP, important mediators of microvessel permeability (Ps), are regulated predominantly by the rates of synthesis and hydrolysis. Phosphodiesterases (PDE) are central to the stimulation or inhibition of hydrolysis of cAMP and cGMP. Our previous studies demonstrated that basal Ps to albumin was greater in adult males (AM) than adult females (AF), juvenile males (JM) and females (JF), but there were no differences among AF, JM, and JF. Therefore, we hypothesized differential PDE expression in AM relative to AF, JM, and JF, resulting in different levels of cAMP and cGMP and consequent difference in basal Ps. In this study we used endothelial cells (EC) derived from rat skeletal muscle microvessels of AM, AF, JM and JF used for Ps studies to detect PDE 1–5 mRNA expression via real time RT‐PCR analysis. We found that mRNA level of PDE1A was 2.5‐fold higher in JM than JF (n=5). PDE2A expression was dependent on maturity while PDE2A levels were higher in juveniles than adults (n=7) in both sexes, and 2‐fold higher in JF (n=6) than JM (n=7). PDE3B was expressed 2.4‐fold higher in JM (n=7) than AM (n=7). PDE4D expression did not differ among the 4 groups of EC. JM (n=5) expressed higher PDE5A than AM (n=6). The findings indicate differential expression of PDE isoforms among the 4 groups of microvessel EC. However, the relationship between PDE expression and levels of cAMP and cGMP remains to be elucidated.

Is Basal Microvascular Permeability Determined by PKA Activity?

Sex-specific differences have been shown to exist in cardiovascular health and disease. Little is known, however, about the role of sex in the regulation of microvascular exchange, particularly microvessel permeability. Previously we have reported that basal permeability (Ps) to albumin of venules isolated from abdominal skeletal muscles was about 2-fold higher in adult male (AM) than adult female (AF), juvenile male (JM), or juvenile female (JF) rats. Further Ps did not differ among AF, JM and JF. It has been demonstrated in frog and rat mesentery that protein kinase A (PKA) played a dominant role in the maintenance of basal microvessel permeability. In this model, the PKA activity was inversely related to Ps. Accordingly, we hypothesized that the high Ps in the AM is the result of lower PKA activity than the other 3 groups and that PKA activity in these 3 groups would not differ. To test this hypothesis, we used primary cultured endothelial cells (EC) derived from the corresponding abdominal skeletal muscles of AM, AF, JM, and JF rats. We quantified the amount of active and inactive PKA in these 4 groups of EC under the resting conditions using immunoblot assay. We found that expression of inactive PKA C-α did not differ among 4 groups of EC (n=4). Of particular interest, PKA activity was not detectable in AF (n=4/4) or AM (n=3/4). In contract, the PKA activity, normalized to JF was 0.19 (n=1/4) and 0.46±0.12 (n=4) for AM and JM, respectively. These findings contrast with the hypothesis of PKA activity being exclusive determinant of basal Ps. The PKA activity data, therefore predict that Ps of AM=AF>>JM >JF in contrast to the observed relationship of AM>AF=JM=JF. Supported by NIH RO1HL078816, HL075186, 1CO6RR017353 and NASA NNJ05HF37G

Transplantation of Autologous Endothelial Cells Induces Angiogenesis

This study examined the feasibility and efficacy of autologous endothelial cell (EC) transplantation using a fibrin matrix in the ischemic myocardium of sheep. Four weeks after placing an ameroid constrictor in the circumflex artery of 12 adult sheep, four animals (EC group) were subjected to EC transplantation. In four others (saline [SAL] group) saline with added inactivated cells was injected and four animals served as controls. Eight weeks after treatment the animals were sacrificed to assess histology and ultrastructure. Eight weeks after injection, ventricular function was markedly improved in the EC transplant group, but had deteriorated in the SAL and control groups. Myocardial blood flow was also increased in the EC group. Histology and electron microscopy revealed extensive neovascularization after EC transplantation and improved myocardial appearance. Heterotopic transplantation of EC within a fibrin matrix enhances neovascularization, increases myocardial blood flow, and improves left ventricular function.

Ambient Pulsatile Pressure Modulates Endothelial Cell Proliferation

Many studies over the last decade have indicated that circulatory forces such as shear stress and cyclic strain can influence the endothelial cell (EC) phenotype. However, very little is known about the in vitro effects of pressure on EC. To study this, cultured bovine aortic EC were grown in custom designed pressure chambers with carefully regulated CO2/air environment. EC were exposed to either atmospheric, static (135 mmHg) or pulsatile pressure (160/110 mmHg). A pulsed pressure frequency of 60 cycles/min was maintained by computer-controlled solenoid valves, placed in series with pressure lines. EC proliferation was determined both by cell count after trypsin release on days 1,3 and 5 and by 3H-thymidine incorporation. By day 5, a significant decrease in cell number occurred in both pressure groups, confirmed by the thymidine studies. No changes were observed in cell morphology and cell viability as assessed by LDH activity studies. To investigate the mechanism of this effect, EC conditioned media from the three pressure conditions were transferred to non-exposed, control EC. Significant cell growth inhibition was demonstrated in the control EC group treated with conditioned media from EC cultured under pulsatile pressure conditions. This finding suggests that EC exposed to pulsatile pressure secrete an autocrine factor with growth inhibitory properties. This effect was not mediated by the growth factors TGFbeta and IL-1 as shown by Northern blot analysis and antibody-neutralization studies.

Anti-angiogenic drug AGM1470 suppresses smooth muscle cell migration induced by endothelial PDGF

To examine the effects of the anti-angiogenic drug AGM1470 on smooth muscle cell (SMC) migration activity stimulated by endothelial cell (EC)-derived mitogen. Study 1; EC's were cultured under pulsatile flow using MCDB151 medium. From the supernatant of these EC dishes we devised two types of conditioned medium; anti-PDGF(+) containing 10 micrograms/ml anti-PDGF antibody, and anti-PDGF(-) containing no antibody. SMC's were cultured using both media. Study 2; EC's were cultured under the same conditions using both types of medium; MCDB151 medium containing 10 ng/ml AGM1470, and MCDB151 medium alone. After the AGM1470 concentration had been adjusted to 10 ng/ml, SMC's were cultured using each medium; AGM-exposed EC and AGM-non-exposed EC. SMC colony spreading distances were measured as an index of mitogenic activity for 4 days. Study 1; the anti-PDGF(-) group showed an apparently greater spreading distance than the anti-PDGF(+) group. Study 2; the AGM-non-exposed EC group showed a significantly greater spreading distance than the AGM-exposed EC group. However, MTT assay revealed no differences in proliferation between the two groups. AGM1470 suppresses the EC production of this PDGF-like mitogen as well as SMC migration activity.

The influence of xenotransplant immunogenicity and immunosuppression on host MHC expression in the rat CNS.

During the early stages following neural transplantation, host immune responses are initiated that are not normally found in the CNS including the induction of major histocompatibility antigens (MHC I and II). Previous laboratory findings have demonstrated prolonged survival of bovine chromaffin cells (BCC) in the rat CNS following transient immunosuppression with cyclosporin A (CSA) providing chromaffin cells are isolated from highly immunogenic passenger cells. To assess the influence of passenger and chromaffin cells on host MHC I and II expression, either BCC, nonchromaffin cell adrenal constituents (NCC), or adrenal medullary endothelial cells (EC) were implanted into the host. At 2 weeks postimplantation, robust BCC survival was obtained in CSA-treated animals. This correlated with low expression of MHC I at the host-graft border and the virtual absence of MHC II. Good BCC survival with reduced MHC I expression only was seen at 6 weeks postimplantation in animals transiently immunosuppressed (4 weeks). In contrast, poor survival was seen in the EC group (even with CSA treatment). In addition, marked MHC I and II expression was found in and around these grafts at 2 weeks, and was particularly intense in EC implanted animals. The results of this study suggest that nonchromaffin passenger cells in BCC preparations, most notably endothelial cells, can induce strong immune responses even in the presence of immunosuppression. Based on MHC staining, removal of these passenger cells can reduce host responses and improve long term survival of xenogeneic chromaffin cells in the CNS.